EMB Agar

Cat. no. G25 EMB Agar, Levine, 15x100mm Plate, 18ml 10 plates/bag
Cat. no. G263 EMB Agar (HHT), 15x100mm Plate, 20ml 10 plates/bag
Cat. no. J21 CLED Agar / EMB Agar, Levine, 15x100mm Biplate, 10ml/10ml 10 plates/bag
Cat. no. J22 Blood Agar / EMB Agar, Levine, 15x100mm Biplate, 10ml/10ml 10 plates/bag
Cat. no. J52 CNA Agar / EMB Agar, Levine, 15x100mm Biplate, 10ml/10ml 10 plates/bag
Cat. no. J58 MacConkey Agar / EMB Agar, Levine, 15x100mm Biplate, 10ml/10ml 10 plates/bag
Cat. no. J88 Rose Agar / EMB Agar, Levine, 15x100mm Biplate,
10ml/10ml
10 plates/bag
Cat. no. P09 EMB Agar, Levine, 15x60mm Contact Plate, 15ml 10 plates/bag

INTENDED USE

Hardy Diagnostics EMB Agar formulations are recommended for use as selective and differential media for the isolation of gram-negative bacilli (including coliform organisms and enteric pathogens) from clinical and nonclinical specimens.

Cat. no. P09 is not intended to be used for the diagnosis of human disease.

SUMMARY

EMB (Eosin Methylene Blue) Agar (HHT) was developed by Holt-Harris and Teague as an alternative to Endo's medium for the isolation of enteric bacilli.(4) Eosin dye and methylene blue were employed to inhibit the growth of gram-positive bacteria; the dyes also serve as differential indicators for the products of fermentation. Lactose and sucrose provide a carbohydrate source. The production of acid from lactose- or sucrose-fermentation results in the eosin-methylene blue dye complex being taken up by bacterial cells to produce a brown to blue-black colony appearance. The HHT formulation provides a clear distinction between colonies of lactose and non-lactose-fermenting microorganisms. However, this formulation does not discriminate between carbohydrate utilization (lactose or sucrose). For example, Yersinia enterocolitica ferments sucrose and not lactose, but will produce the same blue-black colonies as lactose-fermenters.(2,3,5,7-9)

In 1918, Levine simplified the Holt-Harris and Teague formulation by omitting sucrose and doubling the quantity of lactose. The modification facilitated the differentiation of Escherichia coli from Enterobacter aerogenes, and paralleled the reactions of MacConkey Agar for better colony identification.

Historically, EMB Agar, Levine has become the predominant formulation for detecting fecal and non-fecal coliforms. The American Public Health Association recommends its use in the microbiological examination of potable water, waste water, dairy products and foods.(1,11) The USP recommends its use in the performance of Microbial Limit Tests, and the U.S. Food and Drug Administration (FDA) recommends the medium for enumerating Escherichia coli and coliform bacteria.(9,12)

FORMULA

Ingredients per liter of deionized water:*

EMB Agar, Levine:
Pancreatic Digest of Gelatin 10.0gm
Lactose 10.0gm
Dipotassium Phosphate 2.0gm
Eosin Y 0.4gm
Methylene Blue 65.0mg
Agar 15.0gm

Final pH 7.1 +/- 0.2 at 25°C.

EMB Agar (HHT):
Pancreatic Digest of Gelatin 10.0gm
Lactose 5.0gm
Sucrose 5.0gm
Dipotassium Phosphate 2.0gm
Eosin Y 0.4gm
Methylene Blue 65.0mg
Agar 13.5gm

Final pH 7.2 +/- 0.2 at 25°C.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8°C away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

For Cat. nos. G25, G263, J21, J22, J52, J58, and J88.

For Cat. no. P09.

PROCEDURE

Specimen Collection: Consult listed references for information on specimen collection.(1-3,5,8,9,11,12) Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. If there is to be a delay in processing, the specimen should be inoculated onto an appropriate transport media and refrigerated until inoculation.

Method of Use: Allow plates to warm to room temperature. The agar surface should be dry before inoculating. Inoculate and streak the specimen as soon as possible after collection. If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface and streak for isolation with a sterile loop. Incubate plates aerobically at 35-37°C for 18-24 hours and protect from light. Examine plates for colonial morphology. If negative after 24 hours, reincubate an additional 24 hours.

Refer to the document " Contact Plate Media " on the Hardy Diagnostics Technical Document website for more information regarding the method of use for EMB Agar, Levine Contact Plate (Cat. no. P09).

INTERPRETATION OF RESULTS

Following incubation, examine plates for typical colonial morphology.

On EMB Agar, Levine, isolated colonies of lactose-fermenting bacteria appear brown to blue-black in color. Escherichia coli appears as large, blue-black colonies, often with a green metallic sheen. Enterobacter spp. present as brown to blue-black, mucoid colonies with no sheen. Non-lactose-fermenting colonies, such as Shigella spp. and Salmonella spp., appear transparent and colorless.

On EMB Agar (HHT), isolated colonies of lactose- and/or sucrose-fermenting coliforms will produce blue-black colonies with dark centers, often with a green metallic sheen. Other coliforms produce mucoid, pink colonies. Non-lactose- or non-sucrose-fermenting strains, such as Salmonella spp. and Shigella spp., typically present as translucent, amber colored or colorless colonies. Enterococcus faecalis strains may grow as pinpoint or very small colorless colonies.

Consult listed references for further procedures for identification of isolates.(1,3,5,6,8,9)

LIMITATIONS

Some strains of Salmonella and Shigella may fail to grow on EMB Agar.(8)

Some gram-positive bacteria, such as enterococci, staphylococci, and yeast will grow on this medium and usually form pinpoint colonies. Non-pathogenic, non-lactose-fermenting organisms will also grow on this medium. Additional biochemical tests must be performed in order to distinguish these organisms from pathogenic strains.

Serial inoculation may be required to ensure adequate isolation of mixed flora samples.

Some strains of E. coli may fail to produce a characteristic green metallic sheen; consequently, the green metallic sheen is not diagnostic for E. coli.(8)

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, other culture media, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Escherichia coli
ATCC® 25922**
A 24hr 35°C Aerobic Growth; blue-black centered colonies with green metallic sheen
Salmonella enterica
ATCC® 14028
A 24hr 35°C Aerobic Growth; colorless to amber colonies
Enterococcus faecalis
ATCC® 29212**
B 24hr 35°C Aerobic Partial to complete inhibition; pinpoint colonies at 24 hours

** Recommended QC strains for User Quality Control according to the CLSI document M22 when applicable.

USER QUALITY CONTROL

Physical Appearance

EMB Agar, Levine should appear clear, and purple with a green-orange tinge in color. A slight precipitate is also apparent. EMB Agar (HHT) should appear clear to slightly hazy, and dark red to bluish-purple in color.

E. coli growing on EMB Agar, Levine

Escherichia coli (ATCC® 25922) colonies growing on EMB Agar, Levine (Cat. no. G25). Incubated aerobically for 24 hours at 35°C. Shot at an angle to show green sheen.

E. coli growing on EMB Agar, Levine

Escherichia coli (ATCC® 25922) colonies growing on EMB Agar, Levine (Cat. no. G25). Incubated aerobically for 24 hours at 35°C.

S. typhimurium growing on EMB Agar, Levine

Salmonella enterica (ATCC® 14028) colonies growing on EMB Agar, Levine (Cat. no. G25). Incubated aerobically for 24 hours at 35°C.

S. typhimurium growing on EMB Agar, Levine

Salmonella enterica (ATCC® 14028) colonies growing on EMB Agar, Levine (Cat. no. G25). Incubated aerobically for 24 hours at 35°C.

E. faecalis growing on EMB Agar, Levine

Enterococcus faecalis (ATCC® 29212) colonies growing on EMB Agar, Levine (Cat. no. G25). Incubated aerobically for 24 hours at 35°C.

E. coli growing on EMB Agar, Levine

Uninoculated plate of EMB Agar, Levine (Cat. no. G25).

REFERENCES

1. American Public Health Association. Standard Methods for the Examination of Water and Wastewater, APHA, Washington, D.C.

2. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

3. Tille, P.M., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

4. Holt-Harris, J.E. and O. Teague. 1916. A new culture medium for the isolation of Bacillus typhosa from stools. J. Infect. Dis.; 18:596.

5.  Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

6. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology. J.B. Lippincott Company, Philadelphia, PA.

7. MacConkey, A.T. 1905. Lactose-fermenting bacteria in feces. J. Hyg.; 5:333-379.

8. MacFaddin, J.F. 1985. Media for Isolation, Cultivation, Identification, Maintenance of Bacteria , Vol. I. Williams & Wilkins, Baltimore, MD.

9. Versalovic, J., et al. Manual of Clinical Microbiology. American Society for Microbiology, Washington, D.C.

10. Quality Assurance for Commercially Prepared Microbiological Culture Media , M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

11. The Official Compendia of Standards. USP-NF . United States Pharmacopeial Convention, Rockville, MD.

12. U.S. Food and Drug Administration. Bacteriological Analytical Manual. AOAC, Arlington, VA. www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/default.htm

ATCC is a registered trademark of the American Type Culture Collection.

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